Pesquisa de métodos cromatográficos para quantificação e caracterização do certolizumabe pegol

Abstract

Certolizumab pegol (CZP) is a fragment of a recombinant humanized monoclonal antibody (mAb) produced in E. coli, conjugated with polyethylene glycol (PEG) that acts by inhibiting the activity of Tumor Necrosis Factor alpha (TNF-α). CZP has a therapeutic indication for the treatment of chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Size exclusion chromatography (SEC) and Reversed phase liquid chromatography (RP-LC) methods were developed and validated for quantification of CZP in biotechnological products. The CL-EM method was developed on a BioSep-SEC-S3000 chromatographic column (300 x 4.6 mm, 5μm, 290 Å) maintained at 35°C. Mobile phase A consisted of 100mM monobasic sodium phosphate and 200mM sodium chloride pH 7.0 and mobile phase B consisted of Ethanol (95:5, v/v), with a flow of 0.5mL/min and detection by DAD at 214 nm. In the CL-FR method, the analytical condition was determined with a Zorbax 300SB C18 column (4.6 x 150 mm, 3.5 µm), maintained at 80°C, with mobile phase A consisting of 0.1% (v/v) of trifluoroacetic acid (TFA) in water and mobile phase B prepared by propanol:acetonitrile:water:TFA (70:20:9,9:0.1, v/v). Elution took place in a mobile phase concentration gradient at a flow of 1 ml/min and detection by a diode array detector (DAD) at 214nm. CZP was eluted at retention times of 5.6 and 9.0 min, being linear in the concentration range of 1 - 40 mg / mL (r² = 0.9993) and 1 - 40 mg / mL (r² = 0.9997), respectively, for SEC and for RP-LC. The specificity of the methods was investigated by forced degradation studies, interference of formulation excipients and analysis of peak purity. The limits of detection and quantification were 0.14 and 0.41 mg / mL for the SEC method and 0.06 and 0.17 mg / mL for RP-LC. The accuracy means were 100.50 and 99.80 with a bias of 0.85 and 0.82%, respectively, for the SEC and RP-LC methods. The validation of the methods followed the guidelines established in the official guides of the National Health Surveillance Agency (ANVISA) and the International Conference on Harmonization (ICH). In addition, a cell culture bioassay using the MONO-MAC-6 stimulated by lipopolysaccharide (LPS) was studied to evaluate the biological activity of CZP and inhibition of TNF-α release, which proved to be significant. Correlation studies were carried out between the validated methods, aiming to contribute to improve the quality control of the biotechnological product, in order to ensure safety and therapeutic efficacy.